Pharmaceutical agents containing acyclovir, fusaric acid and derivatives thereof

ABSTRACT

Compositions and kits comprising acyclovir and fusaric acid are provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

This invention relates to pharmaceutical agents for the treatment ofviral diseases, and more specifically, to pharmaceutical agentscontaining acyclovir and fusaric acid or derivatives thereof.

BACKGROUND OF THE INVENTION

Acyclovir,2-amino-1,9-dihydro-9-[(2-hydroxyethoxy)metyl]-6H-purin-6-one, has beenused for the inhibition of herpes simplex virus types 1 (HSV-1), 2(HSV-2), and varicella-zoster virus (VZV). Acyclovir is a syntheticpurine nucleoside analogue with in vitro and in vivo inhibitory activityagainst HSV-1, HSV-2 and VZV, which can be found in capsule, tablet,topical and suspension formulations. The following structural formularepresents acyclovir:

The inhibitory activity of acyclovir is highly selective due to itsaffinity for the enzyme thymidine kinase encoded by HSV-1, HSV-2 andVZV. This viral enzyme converts acyclovir into acyclovir monophosphate,a nucleotide analogue. The monophosphate is further converted intodiphosphate by cellular guanylate kinase and into triphosphate by anumber of cellular enzymes. In vitro, acyclovir triphosphate stopsreplication of herpes viral DNA. This is accomplished by: 1) competitiveinhibition of viral DNA polymerase, 2) incorporation into andtermination of the growing viral DNA chain and 3) inactivation of theviral DNA polymerase.

While the use of acyclovir is useful in the treatment of some herpesviruses, it has limitations related to tissue permeability and viralresistance that make its use less desirable. For example, acyclovir isnot effective in topical formulation against herpes labialis and herpesgenitalis. As such, acyclovir may not be used topically to effectivelytreat herpes labialis, herpes genitalis or other viral diseases,disorders, or conditions. Moreover, prodrugs of acyclovir have onlyminimal effects in herpes labialis. For example, the use of denavir totreat herpes labialis reduces the median duration of the illness by 6hours. However, herpes labialis typically lasts in the range of about 6to 7 days. Also, viral strains resistant to acyclovir and its prodrugshave emerged, which further limit its clinical usefulness.

Accordingly, there is a need to provide compositions that may be usedtopically to successfully treat a wide range of viral diseases.

SUMMARY OF THE INVENTION

In overcoming the above disadvantage, it is an object of the inventionto produce compositions, which may be used topically to successfullytreat a wide range of viral diseases.

Accordingly, and in one aspect of the invention, a composition includingat least one nucleoside analogue inhibitor, a pharmaceuticallyacceptable salt, solvate, or prodrug thereof and fusaric acid, aderivative, or pharmaceutically acceptable salt of fusaric acid isprovided.

In a second aspect, a pharmaceutical composition comprising theabove-described composition of the invention and a pharmaceuticallyacceptable carrier is provided.

In a third aspect, a method of treating a viral disease, disorder, orcondition comprising the administration of a pharmaceutically effectivedose of the above-described pharmaceutical composition to a patient isprovided.

In a fourth aspect, a topical preparation is provided that includes atleast one above-described composition of the invention and a liquidvehicle.

In a fifth aspect, a systemic preparation is provided that includes atleast one above-described pharmaceutical composition.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the antiviral effect of fusaric acid on HSV asdetermined by plaque reduction assay.

FIG. 2 illustrates the cytotoxicity profiles of fusaric acid asdetermined by neutral red uptake assay.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been discovered that acomposition can be prepared, which can be used to successfully treat awide range of viral diseases. The composition of the invention comprisesfusaric acid, or any pharmacologically acceptable salt or derivativethereof and at least one nucleoside analogue inhibitor or apharmaceutically acceptable salt, solvate or prodrug thereof. Fusaricacid is the 5-butyl derivative of picolinic acid. As used herein,“nucleoside analogue inhibitor” includes any pharmaceutically acceptablesalt, solvate, or prodrug of a nucleoside analogue inhibitor. Thestructure of fusaric acid and many of its derivatives is represented bythe following structure (formula (I)):

wherein R₁, R₂ and R₄ are selected from the group consisting of apeptide of sixteen amino acids, carboxyl group, methyl group, ethylgroup, propyl group, isopropyl group, butyl group, isobutyl group,secondary butyl group, tertiary butyl group, pentyl group, isopentylgroup, neopentyl group, fluorine, chlorine, bromine, iodine, andhydrogen and R₃ is a butyl group. As used herein, “fusaric acid”includes the above-derivatives thereof.

As used herein, “pharmaceutically acceptable” means it is, within thescope of sound medical judgment, suitable for use in contact with thecells of humans and lower animals without undue toxicity, irritation,allergic response and the like, and is commensurate with a reasonablebenefit/risk ratio. As used herein, “animal” includes humans.“Pharmaceutically acceptable salt” means a relatively non-toxic,inorganic and organic acid addition salt, and base addition salt ofcompounds of the invention. These salts can be prepared in situ duringthe final isolation and purification of the compounds. In particular,acid addition salts can be prepared by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. Exemplary acid addition saltsinclude, but are not limited to the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate,oleate, palmitate, stearate, laurate, borate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates,malonates, salicylates, propionates, methylene-bis-b-hydroxynaphthoates,gentisates, isethionates, di-p-toluoyltartrates, methane-sulphonates,ethanesulphonates, benzenesulphonates, p-toluenesulphonates,cyclohexylsulphamates and quinateslaurylsulphonate salts and the like.Other suitable salts are found in S. M. Berge, et al., “PharmaceuticalSalts,” J. Pharm. Sci., 66: p.1-19 (January 1977), which is incorporatedherein by reference in its entirety.

Base addition salts can also be prepared by separately reacting thepurified compound in its acid form with a suitable organic or inorganicbase and isolating the salt thus formed. Base addition salts includepharmaceutically acceptable metal and amine salts. Suitable metal saltsinclude the sodium, potassium, calcium, barium, zinc, magnesium andaluminum salts. Suitable inorganic base addition salts are prepared frommetal bases which include, but are not limited to sodium hydride, sodiumhydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide,lithium hydroxide, magnesium hydroxide and zinc hydroxide. Suitableamine base addition salts are prepared from amines which have sufficientbasicity to form a stable salt, and preferably include those amineswhich are frequently used in medicinal chemistry because of their lowtoxicity and acceptability for medical use. Suitable amine base additionsalts include, but are not limited to, ethylenediamine,N-methyl-glucamine, lysine, arginine, omithine, choline,N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine,N-benzylphenethylamine, diethylamine, piperazine,tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide,triethylamine, dibenzylamine, ephenamine, dehydroabietylamine,N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, ethylamine, basic aminoacids, e.g., lysine and arginine dicyclohexylamine and the like.

Fusaric acid was recognized in the early 1960's to have activity as anantihypertensive agent in vivo. It also has been shown that fusaric acidis an inhibitor of cancerous cell growth, showing an effect on thegrowth and viability of normal and cancerous cells in tissue culture.U.S. Pat. Nos. 5,767,135 and 6,127,393, herein incorporated by referencein their entireties, show the usefulness of fusaric acid as a potentanti-cancer and anti-viral agent in vivo.

It has now been discovered that when fusaric acid, a derivative, orpharmaceutically acceptable salt thereof is combined with at least onenucleoside analogue inhibitor, pharmaceutically acceptable salt, solvateor prodrug thereof, a synergistic antiviral effect is observed. Forexample, FIGS. 1 and 2 illustrate the antiviral effect of fusaric acidon HSV-1 and HSV-2 as determined by plaque reduction assay, a techniquedescribed in detail below. The 50% effective concentration of fusaricacid was approximately 19 μg/ml for HSV-1 and 18 μg/ml for HSV-2. Theeffective concentration giving 90-100% plaque reduction wasapproximately 30 μg/ml for both HSV-1 and HSV-2.

Fusaric acid is active against HSV-1 and HSV-2 infections. However, asdemonstrated in Example 3 below, the addition of small amounts ofacyclovir or another nucleoside analogue inhibitor to fusaric acid, aderivative or pharmaceutically acceptable salt thereof results inenhanced antiviral effects as compared to the antiviral effects ofeither fusaric acid or acyclovir alone. Specifically, and with referenceto Example 3 below, administration of the compositions of the inventioncomprising both fusaric acid and acyclovir to susceptible cells showabout a 50% reduction of HSV-1 viral activity and about a 40% reductionin HSV-2 viral activity.

The compositions of the invention are prepared by admixing eachcomponent. In embodiments employing additional therapeutic compounds,elevated temperature may be necessary to ensure uniform dispersement ofthe pharmacologically active components through the chosen medium.Generally uniform disbursement of the components occurs at a temperatureof about 45° C. However, one skilled in the art can readily determine,without undue experimentation, the exact conditions necessary to preparethe compositions of the invention, which may vary depending on thespecific composition employed, the nature and presence of othertherapeutic compositions or carriers within the composition of theinvention and the quantity being produced. The compounds of theinvention may alternatively be prepared by other methods well known inthe art.

At least one nucleoside analogue inhibitor, pharmaceutically acceptablesalt, solvate or prodrug thereof must be present in the compositions ofthe invention. Suitable nucleoside analogue inhibitors include, but arenot limited to acyclovir, zidovudine, didanosine, zalcitabine,lamivudine, stavudine, abacavir, combivir, valacyclovir, famciclovir,ganciclovir, ribavirin and denavir. In one embodiment, the compositionsof the inventions are comprised of acyclovir, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof. In a specific embodimentof the invention, the nucleoside analogue inhibitor is an acyclicguanosine analogue. Suitable acyclic guanosine analogues include, butare not limited to acyclovir, valacyclovir, famciclovir, gangciclovirand danavir. As used herein danavir may be optionally referred to aspenciclovir.

Pharmaceutically acceptable prodrugs are those prodrugs of the compoundsthat are within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals with unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compositionsof the invention. The term “prodrug” means compounds that are rapidlytransformed in vivo to yield the parent compound of the above formula,for example by hydrolysis in blood. Functional groups which may berapidly transformed, by metabolic cleavage, in vivo form a class ofgroups reactive with the carboxyl group of the compositions of thisinvention. They include, but are not limited to such groups as alkanoyl(such as acetyl, propionyl, butyryl, and the like), unsubstituted andsubstituted aroyl (such as benzoyl and substituted benzoyl)alkoxycarbonyl (such as ethoxycarbonyl), trialkylsilyl (such astrimethyl- and triethysilyl) monoesters formed with dicarboxylic acids(such as succinyl) and the like. Because of the ease with which themetabolically cleavable groups of the compounds useful according to thisinvention are cleaved in vivo, the compounds bearing such groups act aspro-drugs. The compounds bearing the metabolically cleavable groups havethe advantage that they may exhibit improved bioavailability as a resultof enhanced solubility and/or rate of absorption conferred upon theparent compound by virtue of the presence of metabolically cleavablegroup. A thorough discussion of prodrugs is provided in Glycolamideesters as biolabile prodrugs of carboxylic acid agents: synthesis,stability, bioconversion, and physicochemical properties, Nielsen N M,Bundgaard H., J Pharm Sci. 1988 Apr.;77(4):285-98, which is incorporatedherein by reference.

The relative concentrations of fusaric acid and acyclovir in thecompositions of the invention are dependent upon the specific usage forthe compositions of the invention, e.g. the nature of the disease andthe relative age, condition and size of the patient, as well as themethod of administration and/or carrier employed. Generally, thecompositions of the invention are comprised of in the range of about0.2% to about 50% fusaric acid, a pharmaceutically acceptable salt, orderivative thereof and in the range of about 0.2% to 50% at least onenucleoside analogue inhibitor. In another embodiment, the compositionsof the invention are comprised of in the range of about 1% to about 20%fusaric acid, a pharmaceutically acceptable salt, or derivative thereofand in the range of about 1% to about 20% at least one nucleosideanalogue inhibitor. In yet another embodiment, the compositions of theinvention are comprised of about 5% fusaric acid, a pharmaceuticallyacceptable salt, or derivative thereof and about 5% at least onenucleoside analogue inhibitor. It will be appreciated by those of skillin the art that compositions including a combination of nucleosideanalogue inhibitors are within the scope of the present invention. Itwill also be appreciated by those of skill in the art that compositionshaving concentrations outside of the stated ranges are within the scopeof the present invention.

In one specific embodiment, the composition of the invention comprisesabout 5% acyclovir and about 5% fusaric acid. This particularformulation of the invention has been shown to effectively treat primaryHSV-1 infection in vivo. See Example 4, supra.

The foregoing compositions of the invention may also be mixed with othertherapeutic compounds to form pharmaceutical compositions (with orwithout diluent or carrier) which, when administered, providesimultaneous administration of a combination of active ingredientsresulting in the combination therapy of the invention. Accordingly, andin one aspect of the invention, a pharmaceutical composition is providedcomprising the above-described compositions of the invention and atleast one other therapeutic compound selected from a group consisting ofpharmaceutically acceptable carriers, diluents, for example salinesolutions, adjuvants, excipients, or vehicles, such as preservingagents, fillers, disintegrating agents, wetting agents, emulsifyingagents, suspending agents, sweetening agents, flavoring agents,perfuming agents, antibacterial agents, antifungal agents, lubricatingagents, dispensing agents and any combination thereof. The therapeuticcompound may also be a liquid vehicle, including but not limited to bothan ointment and absorption base. The therapeutic compound employeddepends on the nature of the disease, mode of administration, dosageforms employed, as well as the overall age, size and condition of thepatient. Examples of suspending agents include, but are not limited to,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar and tragacanth and any combination or mixture of thesesubstances.

In certain preferred embodiments, active ingredients necessary incombination therapy may be combined in a single pharmaceuticalcomposition for simultaneous administration.

The choice of vehicle and the content of active substance in the vehicleare also generally determined in accordance with the solubility andchemical properties of the active compound, the particular mode ofadministration and the provisions to be observed in pharmaceuticalpractice. For example, excipients such as lactose, sodium citrate,calcium carbonate, dicalcium phosphate and disintegrating agents such asstarch, alginic acids and certain complex silicates combined withlubricants such as magnesium stearate, sodium lauryl sulfate and talcmay be used for preparing tablets. When aqueous suspensions are usedthey can contain emulsifying agents or agents which facilitatesuspension. Diluents such as sucrose, ethanol, polyethylene glycol,propylene glycol, glycerol and chloroform or mixtures thereof may alsobe used.

Any pharmaceutically acceptable carrier suitable to permit drugadministration may be used. Suitable carriers include, but are notlimited to colorants, flavorants, and other inactive or inertingredients, for example, gelatin, corn starch, lactose, magnesiumstearate, sodium lauryl sulfate, parabens, ink, microcrystallinecellulose, povidone, methyl paraben, preservatives and sodium starchglycolate.

The pharmaceutical compositions of the invention are administered to apatient by any acceptable means, which results in effective antiviralactivity according to the method of the invention. Acceptable means ofadministration include, but are not limited to topical, oral,parenteral, subcutaneous, intravenous, vaginal, intramuscular, systemicand intraperitoneal administration. Accordingly, the pharmaceuticalcompositions of the invention may be administered to a patient, amongother routes of administration, in capsule forms, topical preparationsor intravaginal preparations. As used herein, “patient” includes bothhumans and other animals. It will be appreciated that the preferredroute may vary with, for example, the nature of the disease and thecondition, age and size of the patient.

Topical preparations are provided in accordance with the invention,which comprise the above-described compositions of the invention and aliquid vehicle. Formulations suitable for topical administration areformulations, which are in a form suitable to be administered topicallyto a patient. The formulation may be presented as a topical ointment,salves, powders, sprays and inhalants, gels (water or alcohol based),creams, as is generally known in the art, or incorporated into a matrixbase for application in a patch, which would allow a controlled releaseof compound through the transdermal barrier. When formulated in anointment, the active ingredients may be employed with either aparaffinic or a water-miscible ointment base. Alternatively, the activeingredients may be formulated in a cream with an oil-in-water creambase. Formulations suitable for topical administration in the eyeinclude eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent for theactive ingredient. Formulations suitable for topical administration inthe mouth include lozenges comprising the active ingredient in aflavored basis, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert basis such as gelatin andglycerin, or sucrose and acacia; and mouthwashes comprising the activeingredient in a suitable liquid carrier. The liquid vehicle may be anyliquid vehicle suitable to facilitate the topical application of thecomposition of the invention. Suitable liquid vehicles include but arenot limited to ointment and absorption bases.

The relative concentrations of fusaric acid and nucleoside analogueinhibitor in the topical preparations of the invention are dependentupon the specific usage for the composition of the invention, e.g. thenature of the disease and the relative age, condition and size of thepatient, as well as the method of administration and/or carrieremployed. Generally, the topical preparations of the invention arecomprised of in the range of about 0.2% to about 50% fusaric acid and inthe range of about 0.2% to 50% at least one nucleoside analogueinhibitor. In another embodiment, the topical and intravaginalpreparations of the invention are comprised of in the range of about 1%to about 20% fusaric acid, a pharmaceutically acceptable salt, orderivative thereof and in the range of about 1% to about 20% at leastone nucleoside analogue inhibitor. In yet another embodiment, thetopical and intravaginal preparations of the invention are comprised ofabout 5% fusaric acid, a pharmaceutically acceptable salt, or derivativethereof and about 5% at least one nucleoside analogue inhibitor. It willbe appreciated by those of skill in the art that compositions includinga combination of nucleoside analogue inhibitors are within the scope ofthe present invention. It will also be appreciated by those of skill inthe art that compositions having concentrations outside of the statedranges are within the scope of the present invention.

In one specific embodiment, the topical preparations of the inventioncomprise about 5% acyclovir and about 5% fusaric acid. This particularformulation of the invention has been shown to effectively treat HSV-1in vivo. See Example 4, supra.

If desired, and for more effective distribution, the compounds of theinvention can be microencapsulated in, or attached to, a slow release ortargeted delivery systems such as a biocompatible, biodegradable polymermatrices, e.g. poly(d,1-lactide co-glycolide), liposomes, andmicrospheres and subcutaneously or intramuscularly injected by atechnique called subcutaneous or intramuscular depot to providecontinuous slow release of the compounds for a period of 2 weeks orlonger. The compounds may be sterilized, for example, by filtrationthrough a bacteria retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use.

Formulations suitable for systemic administration are formulations,which are in a form suitable to be administered systemically to apatient. The systemic formulation of the invention include theabove-described compositions of the invention and at least onetherapeutic agent, preferably a liquid vehicle. The formulation ispreferably administered to a patient by injection, includingtransmuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds useful according to the invention areformulated in liquid solutions, e.g., saline solutions, and preferably,in physiologically compatible buffers such as Hank's solution orRinger's solution. In addition, the compounds may be formulated in solidform and redissolved or suspended immediately prior to use. Lyophilizedforms are also included.

Systematic administration also can be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, bile salts and fusaric acidderivatives for transmucosal administration. In addition, detergents maybe used to facilitate permeation. Transmucosal administration may bethrough use of nasal sprays, for example, or suppositories. For oraladministration, the compounds are formulated into conventional oraladministration forms such as capsules, tablets, and tonics.

A therapeutically effective dose of the compositions of the inventionare administered to patients according to the methods of the invention.A therapeutically effective dose is the dosage necessary to provide thedesired physiological change, i.e., to kill the subject virus. Thecompositions of the invention can be prepared in unit dosage form by anyof the methods well known in the art of pharmacy. The specific dosage,however, is dependent upon the means of administration, the generalhealth and physical condition of the patient, as well as the patient'sage and weight, and the specific viral disease, disorder, or conditionsuffered by the patient. The existence of other concurrent treatmentsmay also effect the specific dosage administered to the patient.

The amount of composition of the invention administered to a patient isalso determined by the attending clinicians taking into considerationthe etiology and severity of the disease, the patient's condition, sizeand age, the potency of each component and other factors.

The compositions of the invention may be presented in unit-dose ormultidose containers, for example sealed ampoules and vials withelastomeric stoppers, and may be stored in freeze-dried conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

The compositions of the invention are prepared as described herein or bythe application or adaptation of known methods, by which is meantmethods used heretofore or described in the literature.

The invention also provides kits or single packages combining two ormore active ingredients useful in treating the disease. A kit mayprovide alone or in combination with a pharmaceutically acceptablediluent or carrier, the above-described formula (I) and theabove-described nucleoside analogue inhibitor, pharmaceuticallyacceptable salt, solvate or prodrug thereof, alone, or in combinationwith a diluent or carrier.

The compositions of the invention optionally are supplied as salts.Those salts which are pharmaceutically acceptable are of particularinterest since they are useful in administering the foregoing compoundsfor medical purposes. Salts which are not pharmaceutically acceptableare useful in manufacturing processes, for isolation and purificationpurposes, and in some instances, for use in separating stereoisomericforms of the compounds of this invention. The latter is particularlytrue of amine salts prepared from optically active amines.

All references and patents cited herein are hereby incorporated byreference in their entireties for their relevant teachings. Accordingly,any reference cited herein and not specifically incorporated byreference is, nevertheless, incorporated by reference in its entirety asif part of the specification.

The following examples are illustrative of specific embodiments of theinvention and do not limit the scope of the invention in any way. Ineach example, the plaque reduction technique and cytotoxicity testingprocedures described with reference to Example 1 are used to determinethe percent control of the samples.

EXAMPLE 1 Antiviral Effect of Fusaric Acid on HSV-1 and HSV-2

Samples were prepared for each Example herein by the following method.Stock solutions (200 mg/ml) of fusaric acid were dissolved individuallyin dimethyl sulfoxide (DMSO), aliquoted and stored in a freezer having atemperature of −20° C.

Herpes simplex virus standard strains G (HSV-2) and F (HSV-1) were usedin each Example. Virus input for a 6-well plate was 200 pfu/well. HSVsusceptible cell line, Vero cells, were used in the virus yieldreduction assay. The culture medium for Vero cells is 5% MinimumEssential Medium GIBCO/BRL (MEM) supplemented with 5% fetal bovine serum(200 U/ml penicillin and 200 μg/ml streptomycin).

To examine the efficacy of fusaric acid on HSV-1 and HSV-2, thesusceptible cells were infected with either HSV-1 or HSV-2, thenincubated with fusaric acid at concentrations of 0, 1, 10, 30, 60, and100 μg/ml. The antiviral effect was then determined by plaque reductionassay.

Confluent cells were washed with PBS and infected with either HSV-1 orHSV-2 (200 pfu/well) for 1 hour at 37° C. The cells were tilled every 20minutes. After viral inoculum was removed, infected cells were washedwith PBS and overlaid with 0.5% methylcellulose in culture medium (equalvolume of 1% methylcellulose mixed with 2 times culture medium)containing different doses of testing compounds. The cells wereincubated at 37° C. for 2 days for HSV-2 infection and 3 days for HSV-1.

After plaque size was adequate, the cells were fixed with 20% formalinfor 20 minutes and stained with 0.5% crystal violet for 20 minutes. Thedye was removed by washing with tap water and left to dry in a fumehood. The plaques were then counted. The experiment was repeated.

Mean plaque counts in test wells were compared with mean plaque countsin wells not containing the compound (control wells). The antiviraleffects of fusaric acid on HSV-1 and HSV-2 are summarized in Table 1 andthe antiviral effect of fusaric acid on HSV as determined by plaquereduction assay is shown in FIG. 1.

TABLE 1 Antiviral Effect of Fusaric Acid on HSV-1 and HSV-2 (% ofcontrol) Concentration of Fusaric Acid (μg/ml) 0 1 10 30 60 100 HSV-1100 101 105 0 0(+) 0(++) HSV-2 100 103 101 0 0(+) 0(++) +: ˜20% celldeath observed under microscope. ++: ˜50% cell death observed undermicroscope.

The 50% effective concentrations (ED₅₀) of fusaric acid wasapproximately 19 μg/ml for HSV-1 and 18 μg/ml for HSV-2. EC₉₀₋₁₀₀(effective concentration giving 90-100% plaque reduction) wasapproximately 30 μg/ml for both HSV-1 and HSV-2. Control was calculatedas the mean plaque count in the drug treated wells divided by the meanplaque count in the control wells. The concentrations giving a 50%reduction in the plaque count of the inoculum (EC₅₀) was then calculatedusing computer software.

EXAMPLE 2 Cytotoxicity Profiles of Picolinic Acid and Fusaric Acid

The cytotoxic effects of picolinic acid and fusaric acid were examinedon Vero cells using neutral red dye uptake assay. Confluent Vero cellswere cultured in a medium containing various doses of picolinic acid fortwo days. The testing concentrations were 0, 10, 100, 250, 500 and 1000μg/ml for picolinic acid and 0, 1, 10, 50, 100 and 500 μg/ml for fusaricacid. Culture medium was removed from confluent Vero cells in a 96-wellplate by pump. The cells were then washed once with 200 μl of PBS. 200μl culture medium containing varying concentrations of either picolinicacid or fusaric acid was added to each well. The cells were thenincubated with 0.01% neutral red dye at 37° C. for two days. At the endof incubation, the medium was removed and the cells washed with PBS. 200μl of 0.01% neutral red in PBS was subsequently added to each well andincubated at 37° C. for 30 minutes The dye was then removed and thecells washed twice with 200 μl PBS per well. The dye was extracted byaddition of 200 μl of 50% ethanol/1% glacial acetic acid in PBS to eachwell and incubated at room temperature for 15 minutes with gentleshaking at 120-150 rpm. The absorbance at 550 nm was read on anenzyme-linked immunosorbent assay (ELISA) reader. The toxicity profilesof picolinic acid and fusaric acid on Vero cells analyzed from opticalabsorbance readings are shown in Table 2.

TABLE 2 Cytotoxicity of Picolinic Acid and Fusaric Acid Picolinic acid(μg/ml) 0 10 100 250 500 1000 Toxicity (% of control) 100 105 95 70 5745 Fusaric acid (μg/ml) 0 1 10 50 100 500 Toxicity (% of control) 100101 76 55 49 16

The cytotoxic concentration giving 50% of cell death (CC₅₀) wasapproximately 800 μg/ml for picolinic acid and 100 μg/ml for fusaricacid.

EXAMPLE 3 Drug Combination of Fusaric Acid and Acyclovir

To examine the efficacy of the combination of fusaric acid and acycloviron HSV-1 and HSV-2, the plaque reduction assay technique described inExample 1 was employed. The susceptible cells were infected with HSV-1then incubated with a combination of fusaric acid at concentrations of0, 1, 10, 30 and 60 μg/ml and 1.75% μM acyclovir. Similarly, othersusceptible cells were infected with HSV-2 then incubated with acombination of picolinic acid at concentrations of 0, 1, 10, 30, and 60μg/ml and 6.1 μM acyclovir. A control was used in each instance, whichcomprised neither picolinic acid nor acyclovir. The results aresummarized in Table 3 below.

TABLE 3 Drug Combination of Fusaric Acid with Acyclovir Fusaric acid(μg/ml) 0 0 1 10 30 60 Acyclovir (μM) 0 1.75 1.75 1.75 1.75 1.75 HSV-1(% of control) 100 62.9 45.5 12.7 0.4 0.2 Fusaric acid (μg/ml) 0 0 1 1030 60 Acyclovir (μM) 0 6.1 6.1 6.1 6.1 6.1 HSV-2 (% of control) 100 42.929.7 1.4 0 0

The above results, shown in Table 1, indicate that 10 μg/ml fusaric acidalone had no effect on both HSV-1 and HSV-2. However, when 10 μg/mlfusaric acid is combined with acyclovir, enhanced acyclovir antiviraleffects are realized. Specifically, the results show that the additionof 10 μg/ml of fusaric acid to 1.75 μM acyclovir reduces HSV-1 plaquecounts by about 50% over that seen with 1.75 μM acyclovir alone.

EXAMPLE 4 Drug Combination of Fusaric Acid and Acyclovir to Treat DorsalCutaneous Herpes Simplex Virus I Infection in Guinea Pigs

Compounds within the scope of the present invention exhibit markedpharmacological activities according to tests described herein. Theresults are believed to correlate to pharmacological activity in humansand other animals. The following pharmacological test results aretypical characteristics of compounds of the present invention.

Four compounds were prepared for testing in a guinea pig infected withdorsal cutaneous herpes simplex virus I infection: (1) 5% acyclovir(acv) in a liquid vehicle; (2) 5% fusaric acid in a liquid vehicle; (3)5% acyclovir and 5% fusaric acid in a liquid vehicle; and (4) 20%picolinic acid (pic) having a pH of 3.5 in a liquid vehicle. A controlwas also prepared that comprised 5% acyclovir in polyethylene glycol, aliquid ointment base. Acyclovir ointment is readily commerciallyavailable. The liquid vehicle used was comprised of deionized water(about 47%), petroleum (apx. 22%), mineral oil (apx. 9%), lanolinalcohol (apx. 3%), anhydrous lanolin (apx. 2%) and ceresine wax(140/150) (apx. 7%).

A three-day treatment program was used for all of the compounds tested,including the control. The guinea pigs were prepared in accordance withthe protocol described in M. B. McKeough and S. L. Spruance, AnimalModels of Herpes Skin Infection: Guinea-pig, 209, HANDBOOK OF ANIMALMODELS OF INFECTION, pages 911-918, herein incorporated by reference inits entirety. HSV-1 virus stock (0.035 ml) was introduced to the guineapig through its skin at well-spaced sites of equal size on each side ofthe dorsal midline of a shaved area of the pig by ten activations of asix-pronged spring-loaded vaccination instrument. The day of inoculationwas day 0. Approximately 250 mg of each sample compound was applied twotimes per day for days 1, 2 and 3. In some cases, each sample compoundwas applied up to four times per day. In the case of the sample thatincluded both acyclovir and fusaric acid, the sample was applied twotimes per day because of skin irritation. To better see the lesions, adepilatory cream was used to remove regrown hair on the dorsum on day 4.Lesions were then counted and lesions sizes were measured usingpictures.

The animals were then sacrificed and the full-thickness skin fromdifferent infected treatment areas of the guinea pig were removed. Theskin from each of the infected treatment areas was placed into 15milliliters (mils) of tissue culture medium with 2% Fetal Bovine Serumin an ice bath. The samples were then homogenized in a Stomacher 80Biomaster lab blender, commercially available from Seward Company,London, United Kingdom. Debris was pelleted by centrifugation and thesupernatants collected and frozen at −70° C. until assay for infectivityby plaque formation in VERO cells was performed. The results aresummarized in Tables 4a and 4b below.

TABLE 4a Efficacy Analysis 5% 20% ACV + Pic 5% % 5% % 5% % Acid % ACV/Diff Fusaric Diff Fusaric Diff PH Diff Veh (p) Veh Acid (p) Veh Acid (p)Veh 3.5 (p) Veh Lesion Number² mean 49 6¹ 52 42 19 52 25 52 52 51 — 51sd 20 (0.5)³  7 12 (.13) 7 9 (.02)  7 20 (.96) 9 n 12 12 7 12 7 12 11 12median 51.5 53.0 41.0 53.0 25.0 53.0 56.0 52.5 Total Lesion Area mm2mean 212 20 264 153 41 258 79 71 266 230 18 281 sd 46 (.005) 46 63 (.02)43 31 (.03)  47 56 (.02) 43 n 12 12 7 12 6 12 11 12 median 204 273 166270 87 279 220 277 Titer log (pfu/ml) mean 4.04 46 4.31 4.03 75 4.643.08 97 4.54 4.43 41 4.66 sd .41 (.03) .43 .60 (.02) .35 .45 (.002) 42.39 (.08) .40 n 12 12 12 12 12 12 12 12 median 4.20 4.36 3.98 4.59 3.184.63 4.40 4.64 ¹. Percent differences between mean lesion severity atdrug-treated sites compared to the vehicle-treated sites are shown. Apositive value indicates a reduction in lesion severity for the testcompound. ². Day 0 is the day of infection. 5% ACV in NVL vehicle, 5%Fusaric Acid, 5% ACV + 5% Fusaric Acid, 50% Picolinic Acid and 5%ACV/PEG were used 2x/day on Days 1, 2 and 3. Efficacy measurements weredone on the morning of Day 4. ³. For statistical analysis, paired datawere evaluated by the Wilcoxon signed rank test, utilizing the log meansof the drug and vehicle results. ⁴. nd = not done.

TABLE 4b Efficacy Analysis 5% ACV/ % Diff PEG (p) polyethyleneglycolLesion Number² mean 51   4¹ 53 sd 9 (.44)³ 7 n 12 12 median 55.0 54.5Total Lesion Area mm2 mean 183 24 241 sd 43  (.005) 44 n 12 12 median179 242 Titer log (pfu/ml) mean 3.98 49 4.27 sd .58 (.01) .56 n 12 12median 4.08 4.20 ¹Percent differences between mean lesion severity atdrug-treated sites compared to the vehicle-treated sites are shown. Apositive value indicates a reduction in lesion severity for the testcompound. ²Day 0 is the day of infection. 5% ACV in NVL vehicle, 5%Fusaric Acid, 5% ACV + 5% Fusaric Acid, 50% Picolinic Acid and 5%ACV/PEG were used 2x/day on Days 1, 2 and 3. Efficacy measurements weredone on the morning of Day 4. ³For statistical analysis, paired datawere evaluated by the Wilcoxon signed rank test, utilizing the log meansof the drug and vehicle results.

The results indicate that compositions comprising 5% acyclovir as theactive agent reduce the number of lesions present by 6%, the totallesion area by 20% and the virus titer by 46%. Compositions comprised of5% fusaric acid as the active agent significantly reduced the number oflesions present by 19% total lesion area by 41% and virus titer by 75%.Compositions comprised of 5% acyclovir and 5% fusaric acid as the activeagents statistically significantly reduced lesion number by 52%, totallesion area by 71% and virus titer by 97%. Compositions that included20% picolinic acid as the active agent significantly reduced the totallesion area by 18% and the virus titer by 41% and did not show a trendin reducing lesion number.

The Mann-Whitney rank sum test was used to compare the efficacy of thesamples. The Mann-Whitney rank sum test is well known to those skilledin the art. The results showed that the compositions that included 5%acyclovir and 5% fusaric acid as the active agents were better than thecompositions that comprised 5% acyclovir, 5% fusaric acid and 20%picolinic acid independently as the active agents in all threeparameters tested. Further, only the combination of acyclovir andfusaric acid was able to affect all three parameters in a statisticallysignificant manner.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results are obtained. Asvarious changes could be made in the above compounds and methods withoutdeparting from the scope of the invention, it is intended that allmatters contained in the above description shall be interpreted asillustrative and not in a limiting sense.

We claim:
 1. A composition comprising about 1.75 μM of acyclovir andabout 1 μg/ml of fusaric acid.
 2. A kit comprising about 1.75 μM ofacyclovir and about 1 μg/ml of fusaric acid.
 3. A composition comprisingabout 1.75 μM of acyclovir and about 10 μg/ml of fusaric acid.
 4. Acomposition comprising about 6.1 μM of acyclovir and about 1 μg/ml offusaric acid.
 5. A composition comprising about 1.75 μM of acyclovir andabout 1 μg/ml to about 10 μg/ml of fusaric acid.
 6. A compositioncomprising about 6.1 μM of acyclovir and about 1 μg/ml to about 10 μg/mlof fusaric acid.
 7. A composition comprising about 6.1 μM of acyclovirand about 10 μg/ml of fusaric acid.
 8. A kit comprising about 6.1 μM ofacyclovir and about 1 μg/ml of fusaric acid.
 9. A kit comprising about6.1 μM of acyclovir and about 10 μg/ml of fusaric acid.
 10. A kitcomprising about 1.75 μM of acyclovir and about 1 μg/ml of fusaric acid.11. A kit comprising about 1.75 μM of acyclovir and about 10 μg/ml offusaric acid.
 12. A kit comprising about 1.75 μM of acyclovir and about1 μg/ml to about 10 μg/ml of fusaric acid.
 13. A kit comprising about6.1 μM of acyclovir and about 1 μg/ml to about 10 μg/ml of fusaric acid.